Treatment of arrhythmias via inhibition of a multifunctional calcium/calmodulin-dependent protein kinase

ABSTRACT

This invention provides a method for treating or preventing arrhythmias in a human subject comprising the administration of an effective amount of a calcium/calmodulin-dependent protein kinase inhibitor. Also provided are pharmaceutical compositions comprising a calcium/calmodulin-dependent protein kinase inhibitor and a pharmaceutically acceptable carrier and methods for identifying agents useful for the treatment of arrhythmias.

This application is a division of U.S. Ser. No. 09/016,145 filed Jan.30, 1998 now U.S. Pat. No. 6,518,245, incorporated by reference herein.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates to methods for the treatment ofarrhythmias by inhibition of a multifunctionalcalcium/calmodulin-dependent protein kinase (CaM kinase), pharmaceuticalcompositions useful in such treatments, and methods for identifying newagents useful for such treatments.

b) Description of Related Art

Arrhythmias are a leading cause of cardiac-related death in the UnitedStates. Prolongation of the cardiac action potential is an importantpredisposing condition for these arrhythmias. Many antiarrhythmic drugsdirectly prolong the action potential duration and so may furthercontribute to these arrhythmias (i.e. the proarrhythmic effects ofantiarrhythmic drugs). Despite their cost, implantable cardiacdefibrillators (ICDs) have become the treatment of choice forarrhythmias. In order to prevent painful shocks ˜50% of patients withICDs require additional treatment with antiarrhythmic drugs. Thus thereis an important need to develop better antiarrhythmic drug therapies.

Early afterdepolarizations (EADs) are depolarizing oscillations in theaction potential (AP) that occur during repolarization. One cause ofEADs is inward L-type Ca2+ current (ICa). ICa is present at cellmembrane potentials (Vm) within the window of ICa steady stateactivation and inactivation overlap. ICa steady state activation andinactivation overlap occur during action potential repolarization.Prolongation of action potential repolarization may increase the timethat the Vm is in the window current range for ICa and thus thelikelihood of EADs. EADs are important because they are one probablecause of lethal arrhythmias associated with long QT intervals includingtorsade de pointes. A long QT interval reflects prolonged actionpotential repolarization in ventricular myocardium and is due to a widevariety of conditions including bradycardia and hypokalemia. Oneimportant cause of long QT intervals are antiarrhythmic drugs and theventricular proarrhythmic effects of many antiarrhythmic agents are dueto QT interval prolongation.

Intracellular Ca²⁺ increases simultaneously with EADs in isolatedventricular myocytes (De Ferrari et al. (1995) Circ 91:2510-2515).Elevation of intracellular Ca²⁺ ([Ca²⁺]_(i)) has complex effects onI_(Ca) including indirect enhancement through a multifunctionalCa²⁺/calmodulin—dependent protein kinase II pathway (Anderson et al.(1994) Circ Res 75:854-861) and direct inactivation. The net effect ofelevated [Ca²⁺]_(i) in rabbit ventricular myocytes following flashphotolysis of the photolabile Ca²⁺ chelator Nitr-5 is 40-50%augmentation of peak I_(Ca) that is mediated by a multifunctionalCa²⁺/calmodulin-dependent protein kinase II, hereafter referred to asCaM kinase II. CaM kinase II, like other multifunctionalCa²⁺/calmodulin-dependent protein kinases, is an ubiquitous serinethreonine kinase that is activated when Ca²⁺ is bound to the Ca²⁺binding protein calmodulin. Once activated by Ca²⁺/calmodulin, CaMkinase II activation may be sustained by intersubunit enzymeautophosphorylation that confers Ca²⁺-independent activity, allowing forits activity to persist during the long diastolic intervals associatedwith QT interval prolongation and torsade de pointes. ThisCa²⁺-independent activity is enhanced by long stimulating pulses (DeKoninck and Schulman (1998) Science 279:227-230), as occur withprolonged action potential repolarization. It is possible that EADscaused by I_(Ca) may be enhanced by increased [Ca²⁺]_(i) through the CaMkinase II pathway. However, not all EADs are due to I_(Ca), and EADs canoccur in conditions adverse to CaM kinase activity such as enhanced[Ca²⁺]_(i) buffering.

Delayed afterdepolarizations (DADs) are another cause of ventriculararrhythmias associated with intracellular calcium overload. DADs arecaused by an inward current that follows completion of action potentialrepolarization. This inward current is a marker of intracellular calciumoverload (Thandroyen et al. (1991) Circ Res 69:810-819). Intracellularcalcium overload is a central feature of many ventricular arrhythmiasoccuring during ischemia (Lee et al. (1988) Circ 78:1047-1059) includingventricular fibrillation.

Inhibition of CaM kinase activity can be used to test for a facilitatoryrole of CaM kinase in EADs and DADs. There are several methods forblocking CaM kinase activity. Synthetic pseudo-substrate peptideinhibitors of CaM kinase provide a specific approach to CaM kinaseinhibition and have been used in a variety of cell types includingventricular myocytes (Braun et al. (1995) J. Physiol 488:37-55). Thepeptide sequence KKALHRQEAVDCL (SEQ ID NO: 1), like other similarpeptides, was found to inhibit CaM kinase in a highly specific manner.SEQ ID NO: 1 is a much less efficient inhibitor of both protein kinase A(PKA) and protein kinase C (PKC), with an IC₅₀ value of at least 500μmol/l for each. Myristoylated inhibitory peptides are cell membranepermeant and thus could also be effective when added extracellularly.

A variety of cell-membrane permeant organic CaM kinase and calmodulininhibitors are available and widely used (Braun et al. (1995) Ann RevPhysiol 57:417-445). A principle disadvantage for these inhibitors asexperimental agents is that many of them directly block I_(Ca). However,direct I_(Ca) blockade by the CaM kinase inhibitor KN-93 has not beenpreviously reported. KN-93(2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)is a methoxybenzene sulfonamide derivative that competitively inhibitscalmodulin binding to CaM kinase with a reported K_(i) of 0.37 μmol/l.KN-93 has been shown to inhibit CaM kinase-dependent processes in PC12hcells, fibroblasts, and gastric parietal cells. There are four isoformgroups of CaM kinase II (α,β,γ,δ) and the δ_(B) and δ_(C) isoforms havebeen identified in myocardium. The catalytic and regulatory domains inCaM kinase are highly conserved in all known CaM kinase isoforms soinhibitors that interact with either of these domains are expected towork in all cell types including cardiac. KN-92(2-N-(4-methoxybenzenesulfonyl)-amino-N-(4-chlorocinnamyl)N-methylbenzylamine)is a congener of KN-93 without CaM kinase inhibitory activity and isused as an experimental control. Direct I_(Ca) blockade by KN-92 hasalso not been previously reported, and neither KN-93 nor KN-92 haveappreciable effects on other serine threonine kinases such as proteinkinase A (PKA) or protein kinase C (PKC).

Two general approaches are currently used to suppress ventriculararrhythmias due to action potential prolongation, in addition to ICDimplantation. The first is to shorten the action potential usingantiarrhythmic agents (e.g. mexilitine, pinacidil) or to increase theheart rate using artificial pacing or the β-adrenergic agentisoproterenol. The second is to indirectly suppress protein kinase A(PKA), which enhances L-type calcium current and calcium release fromthe sarcoplasmic reticulum, through left stellate ganglionectomy or withβ-adrenergic blocking drugs. Neither of these approaches is broadlyapplicable for two reasons: 1) action potential duration is governed bya number of different ionic currents and it is not typically known whichcurrent is critical in a given patient. Furthermore, specificantiarrhythmic drugs are not available for modification of many of theseionic currents. In addition, not all causes of action potentialprolongation respond to pacing by action potential shortening.Isoproterenol also does not always shorten the action potential, canitself be arrhythmogenic, may cause ischemia, and can only be used in anacute setting. 2) At present, suppression of PKA is accomplishedindirectly by stellate gangionectomy or by β-adrenergic antagonists.β-adrenergic antagonists are effective in reducing death fromarrhythmias, but application is limited by the fact that these agentsweaken the force of heart muscle contraction.

Ventricular arrhythmias due to ischemia and intracellular calciumoverload are generally treated with revascularization (i.e. coronaryartery angioplasty or coronary artery bypass surgery), β-adrenergicantagonists, and class III antiarrhythmic medications (e.g. sotalol andamiodarone), in addition to ICDs. Unfortunately revascularization isoften incomplete and the recurrence rate of ventricular fibrillation issignificant. Class III antiarrhythmic agents may be proarrhythmic bycausing excessive action potential prolongation or be associated withuse-limiting toxicity (e.g. amiodarone). Beta-adrenergic antagonist useis also limited as previously discussed (above).

Atrial fibrillation is associated with significant morbidity andmortality from stroke and heart failure. Atrial fibrillation can becaused by DADs. Maintenance of atrial fibrillation is favored byintracellular calcium dependent processes. (Tielenian et al. (1997) Circ95:1945-1953) Present conventional therapies center aroundanticoagulation (for prevention of stroke), ventricular rate control,and antiarrhythmic agents. Currently available antiarrhythmic agentsonly succeed in maintaining sinus rhythm in approximately fifty percentof patients per year. Recent experimental therapies include artificialpacing systems and atrial ICDs.

CaM kinase inhibition may be superior to previous antiarrhythmicstrategies because CaM kinase has characteristics that may allow it tofunction as a proarrhythmic positive feedback effector for EADs andDADs, and thus play a more central role in EAD and DAD induction thanother effectors, such as PKA. L-type calcium current increasesintracellular calcium directly by transmembrane calcium flux andindirectly through calcium-induced release of calcium from thesarcoplasmic reticulum. Increased intracellular calcium results inenhanced CaM kinase activity to further favor EADs during actionpotential prolongation. In contrast, PKA activity is not enhanced byincreased intracellular calcium. Direct CaM kinase inhibitors areavailable (above) and CaM kinase inhibition does not reduce the strengthof contraction in isolated hearts, suggesting it may be applicable inpatients who do not tolerate β-adrenergic antagonists. Recentexperiments using genetically engineered mice lacking neuronal CaMkinase isoforms suggest that systemic CaM kinase inhibition will notresult in intolerable side effects. It is likely that redundant CaMkinase isoforms or other signal transduction mechanisms partiallycompensate for the inactivity of one CaM kinase subtype, so the actualeffects of knockout or other inhibition strategies are less deleteriousthan anticipated. Thus, CaM kinase inhibition may be highly beneficialas a treatment for arrhythmias related to excessive action potentialprolongation and EADs and for arrhythmias related to intracellularcalcium overload such as atrial and ventricular fibrillation.

Action potential prolongation favors increased L-type calcium current,and this current is the likely proximate cause of the arrhythmias, butbecause calcium is essential for cardiac muscle function, directblockade of L-type calcium current is not a viable antiarrhythmicstrategy. L-type Ca²⁺ current inhibitors have not been found to behighly effective antiarrhythmic agents for atrial and ventricularfibrillation as well as most types of ventricular tachycardia. Lack ofventricular antiarrhythmic efficacy for I_(Ca) antagonists at clinicallytolerated doses may be because the amount of I_(Ca) inhibition isinsufficient to prevent or terminate most EADs. Combination of a I_(Ca)antagonist with a CaM kinase inhibitor may be effective, however.

SUMMARY OF THE INVENTION

The present invention provides methods for treating and preventingarrhythmias in a human subject through the administration of amultifunctional calcium/calmodulin-dependent protein kinase inhibitor tothe human in an amount sufficient to suppress earlyafterdepolarizations, delayed afterdepolarizations, or intracellularcalcium overload.

Also provided are pharmaceutical compositions comprising amultifunctional calcium/calmodulin-dependent protein kinase inhibitorand a pharmaceutically acceptable excipient.

Another aspect of the invention provides for methods of identifyingagents useful in the treatment of arrhythmias and provides feasiblestrategies for the development of a therapeutic, antiarrhythmic drugwhich can be applied systemically.

In another embodiment, the present invention provides for methods oftreating and preventing arrhythmias in which a multifunctionalcalcium/calmodulin-dependent protein kinase inhibitor is administered incombination with a second antiarrhythmic agent in order to increasesafety and efficacy of the treatment.

In another embodiment, the present invention provides for methods oftreating and preventing arrhythmias in which the administration of amultifunctional calcium/calmodulindependent protein kinase inhibitor iscombined with treatment of the patient with an antiarrhythmic device.

The present invention also provides for the administration of amultifunctional calcium/calmodulin-dependent protein kinase inhibitor incombination with a second antiarrhythmic agent to block theproarrhythmic effects due to action potential prolongation caused by thesecond antiarrhythmic agent.

DESCRIPTION OF THE FIGURES

FIG. 1. (A) Experimental design for early afterdepolarizations (EAD)induced by clofilium in an isolated rabbit heart pretreated with, theinactive analog, KN-92. Data tracings show (from top to bottom)monophasic action potentials (MAP) and left ventricular (LV) pressure.The horizontal lines to the left of the LV pressure tracings (top tobottom) indicate 100 mm Hg and 0 mm Hg levels and 850 ms for panels Aand B. All experiments with isolated hearts consisted of 3 periods. Afourth period (experimental period 4) was only added in hearts withEADs. Experimental periods are labeled 1-4 (top of page for both panelsA and B). Experimental period 1 measurements represent control andfollow >10 min of stabilization. Experimental period 2 measurements aretaken 10 min after addition of the inactive KN-93 analog, KN-92 (0.5μmole/l). Period 3 measurements follow addition of clofilium (7.5μmole/l) and are taken at EAD initiation or from the longest actionpotential duration recorded over 30 min. Multiple EADs are seen as 2oscillations during repolarization on the MAP tracings in experimentalperiod 3. Secondary elevations in LV pressure coinciding with EADs arealso seen in the LV pressure tracing in experimental period 3.Experimental period 4 measurements follow EAD termination by addition ofnifedipine (10 μmole/l) (shown above) or Cd²⁺ (200-500 μmole/l) (datanot shown). The horizontal bars indicate the experimental conditions forboth panels A and B. (B) Experimental design for isolated rabbit heartstreated with CaM kinase inhibitor KN-93. Arrangement of data tracings isthe same as in FIG. 1A (above). Experimental periods are also the sameas in FIG. 1A (above) except the active CaM kinase inhibitor KN-93 (0.5μmole/l) was added 10 min prior to period 2 measurements. Period 4 wasomitted because EADs did not appear within 30 min after addition ofclofilium. (C) Continuous record of EAD termination by nifedipine (10μM) in a heart paced at a cycle length of 1500 ms. Data tracings showMAP with single EADs (top) and LV pressure (bottom). The horizontallines to the left of the LV pressure tracings indicate 50 mm Hg and 0 mmHg pressure levels (top to bottom). EAD termination occurs prior to thefirst discernible decrease (i.e. >10% below baseline) in LV pressure.The vertical arrow marks the first beat of EAD termination.

FIG. 2. Left ventricular developed pressure (LVDP, panel A) andinterbeat intervals (panel B) in isolated rabbit hearts. LVDP is definedas the peak systolic pressure minus the end diastolic pressure. Numerals1-4 correspond to the experimental periods illustrated in FIGS. 1A andB. The data set shown in panels A and B is clofilium (7.5 μmole/l,n=21); the CaM kinase inhibitor KN-93 (0.5 μmole/l, n=10, open circles)or the inactive analog KN-92 (0.5 μmole/l, n=11, filled circles); EADtermination, nifedipine (10 μmole/l, n=7) or Cd²⁺ (200-500 μmole/l,n=4). Experimental period 4 only includes data from hearts demonstratingEADs; in this last group, n=11 because 3 MAP recordings were lost uponEAD termination. LVDP at experimental period 4 is taken from the firstbeat after EAD termination as shown in FIG. 1C. A. Hearts pre-treatedwith KN-93 failed to increase LVDP following clofilium treatment (panelA, experimental period 3). LVDP did increase significantly afterclofilium addition in hearts treated with the inactive analog KN-92:*p=0.014 compared with period 1; **p=0.002 compared with KN-92 treatedhearts in period 3. B. Interbeat intervals increased compared withcontrol period intervals both in hearts treated with KN-93 (**p=0.02)and in hearts treated with the inactive analog KN-92 (*p=0.049). Nosignificant differences in interbeat intervals were present betweenhearts treated with the CaM kinase inhibitor KN-93 or the inactiveanalog KN-92.

FIG. 3. Monophasic action potential durations (MAP) at 50% (APD₅₀,circles) and 90% (APD₉₀, squares) repolarization to baseline in isolatedrabbit hearts. Numerals 1-4 correspond to the experimental periodsillustrated in FIGS. 1A and B. Filled symbols are for hearts pre-treatedwith the inactive analog KN-92. Open symbols are for hearts pre-treatedwith the CaM kinase inhibitor KN-93. The data set is the same as in FIG.2. *p=0.004 and **p<0.001 compared with corresponding APDs in period 1.No significant differences in MAP durations were present between heartspretreated with the CaM kinase inhibitor KN-93 or the inactive analogKN-92. MAP durations were not significantly different during earlyafterdepolarizations (EAD) and the first beat after EAD termination bynifedipine (10 μmole/l) or Cd²⁺ (200-500 μmole/l).

FIG. 4. Percent calcium-independent CaM kinase activity in isolatedrabbit hearts. Maximal (i.e. Ca²⁺/calmodulin-dependent) andCa²⁺-independent CaM kinase activities were assayed from leftventricular (LV) tissue homogenates as described in Example 5,Experimental Methods. All hearts were initially isolated under controlconditions. A stabilization period of ≧10 min was used for each isolatedheart to ensure the absence of EADs, as indicated by monophasic actionpotentials (MAPs) recorded by a catheter positioned over the LVepicardium. To assay for CaM kinase activity, a left ventricularepicardial slice (1-2 g tissue) was cut from hearts in each of the threegroups as follows: 1) Control (n=5), slice removed after ≧10 minstabilization period; 2) Clofilium (n=5), slice removed after additionof 7.5 μmole/l clofilium and ≧10 min of EADS as verified by MAPrecordings; 3) KN-93, Clofilium (n=6), slice removed ≧10 min afteraddition of 7.5 □mole/l clofilium in hearts pretreated with 0.5 μmole/lKN-93. * p=0.015 compared to both the Control group and the KN-93,Clofilium group.

FIG. 5. Effects of KN-93 and KN-92 on CaM kinase activity in vitro. CaMkinase activity in left ventricular tissue homogenate was assayed asdescribed in Example 5, Experimental Methods. The plot shows thatincreasing concentrations of KN-93, but not the inactive analog KN-92,produce direct inhibition of cardiac CaM kinase activity. The solid linerepresents a fit to the KN-93 data using the Hill equation. The dashedline shows the expected inhibition of CaM kinase activity afteraccounting for the concentration of added calmodulin in the assay (150nmole/l), given the competitive interaction between KN-93 and calmodulinwith CaM kinase.(Sumi et al. (1991) Biochem Biophys Res commun181:968-975) The K_(i) value for inhibition by KN-93 calculated from thedashed curve is 2.58 μmole/l.

FIG. 6. KN-93 and KN-92 effects on L-type Ca²⁺ current (I_(Ca)). (A).Peak steady state current voltage (I-V) relationship for I_(Ca) duringcontrol conditions (filled squares) and following addition of theinactive agent KN-92 (0.5 μmol/l filled circles and 1.0 μmol/l filleddiamonds) to the cell bath. The inset shows superimposed raw currentsand the command voltage step (300 ms) from the ventricular myocyte usedto construct the I-V relationship. The horizontal bar marks the zerocurrent level. (B). Tracings are laid out as in A. (above) except theCaM kinase inhibitor KN-93 was added to the cell bath (0.5 umol/l opencircles and 1.0 umol/l open diamonds). (C). Dose response relationshipsfor peak steady state I_(Ca), recorded during command voltage steps froma holding potential of −80 mV to a test potential of 0 mV, and the CaMkinase inhibitor KN-93 (open circles) or the inactive analog KN-92(filled circles). Data are expressed as a fraction of control I_(Ca).The total number of cells studied during each plotted measurement isgiven in parentheses. KN-93 and KN-92 were equipotent peak steady stateI_(Ca) inhibitors at the concentration used to suppress earlyafterdeploarizations in this study, but KN-93 was a more effectiveI_(Ca) inhibitor at 1.0 μmol/l. (D). Time dependence of I_(Ca) recoveryfrom inactivation. Peak I_(Ca) was elicited from a holding potential of−80 mV to a test potential of 0 mV for 300 ms (P1). Progressively longerintervals (10-2500 ms) were inserted between P1 and a second pulse (P2).Peak I_(Ca) during P2 was expressed as a fraction of peak I_(Ca) duringP1. Data are plotted for control conditions (filled squares) in thepresence of 0.5 μmol/l KN-92 (filled circles) or 0.5 μmol/l KN-93 (opencircles).

FIG. 7. CaM kinase inhibition suppresses pacing induced inward current.Isolated heart cells were paced (0.5 Hz, T=32° C.) using a prolongedaction potential voltage clamp command wave form. (A) A prolonged actionpotential was digitized and used as a voltage clamp command waveform forcurrent tracings below. Command membrane potential is on the ordinateand time (for panels A-C) is on the abscissa (B) Inward currentsdeveloped after completion of the command wave form in 5/6 cellsdialyzed with a control peptide (SEQ ID NO: 2, 20 μmole/l) devoid of CaMkinase inhibitory activity. Cell membrane currents are referenced on theordinate for both panels B and C. (C) Inward currents did not develop inany (0/5) cells dialyzed with the active CaM kinase inhibitory peptide(SEQ ID NO: 1, 20 μmole/l). In addition, ryanodine (10 μmole/l), ablocker of intracellular calcium release, also prevented inward currentdevelopment in 2/2 cells tested.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered a method for treating arrhythmias in which amultifunctional calcium/calmodulin-dependent protein kinase inhibitor isadministered to the patient in an amount sufficient to suppress earlyafterdepolarizations, delayed afterdepolarizations, or intracellularcalcium overload. The present invention also provides for the preventionof the occurrence of arrhythmias through the administration of amultifunctional calcium/calmodulin-dependent protein kinase inhibitor tothe patient patient in an amount sufficient to suppress earlyafterdepolarizations, delayed afterdepolarizations, or intracellularcalcium overload.

The term “CaM kinase” is used herein to refer to any member of themultifunctional calcium/calmodulin-dependent protein kinase family ingeneral, whereas the term “CaM kinase II” is used herein only to referto the multifunctional calcium/calmodulin-dependent protein kinase II(and its isoforms).

An “inhibitor” of CaM kinase is a compound capable of decreasing theactivity of the enzyme (generally via noncovalent binding of theinhibitor to the enzyme). The inhibitor may provide reversibleinhibition. Reversible inhibition can consist of competitive inhibition,noncompetitive inhibition, uncompetitive inhibition, or mixedinhibition. An “inhibitor” of CaM kinase can also be an irreversibleinhibitor or it can be a suicide inhibitor.

In a preferred embodiment of the invention, an inhibitor of amultifunctional calcium/calmodulin-dependent protein kinase II is usedin the treatment and prevention of arrhythmias. However, the use ofcompounds which inhibit other members of the family of multifunctionalcalcium/calmodulin-dependent protein kinases is also provided for by thepresent invention. The family of multifunctionalcalcium/calmodulin-dependent protein kinases includes CaM kinase I, CaMkinase II, and CaM kinase IV. Each of these members also additionallyexists in a multitude of isoforms. An inhibitor of one of themultifunctional calcium/calmodulin-dependent protein kinases isgenerally understood by one skilled in the art to be likely to inhibitthe other members of the family as well, due to structural conservationamongst the types (Enslen et al. (1994) J. Biol. Chem. 269:15520-15527).

In another embodiment of the present invention, isoform-specificinhibitors could be used as agents with enhanced specificity in theprevention and treatment of arrhythmias (Braun et al. (1995) J. Physiol488:37-55). In still another embodiment, organ-specific CaM kinaseinhibtors could be used. Cardiac specificity of a multifunctionalcalcium/calmodulin-dependent protein kinase may be achieved by combininga CaM kinase inhibitor with cardiac cell specific epitope recognitionsequences. The use of organ or isoform specific inhibitors may helpensure efficacy and the safety of systemic delivery of the drug.

Existing CaM kinase inhibitors such as KN-93 and any of theinhibitory-peptides such as KKALHRQEAVDCL (SEQ ID NO: 1) can be used inthe methods of treating arrhythmias described herein. Both inhibitorsact competitively, but at distinct sites on the kinase. KN-93 binds andprevent activation by calmodulin, whereas SEQ ID NO: 1 binds to thecatalytic site, preventing interaction with substrate molecules. Othergeneral, organ-specific, or isoform-specific CaM kinase inhibitors mayalso be used in the treatment of arrhythmia as provided for by thepresent invention.

Administration of the inhibitor to the patient may be accomplishedthrough any one of a variety of methods known to those skilled in theart. In a preferred embodiment, the inhibitor is delivered systemically.In a further preferred embodiment, the CaM kinase inhibitor is deliveredintravenously. Other possible methods of delivery include, but are notlimited to oral delivery, bolus injection or continuous parenteral pumpinfusion, cutaneous patch or cutaneous iontophoretic delivery, andintracoronary or intrapericardial delivery, or gene transfer therapy, asrecently described by Griffith et al. (Griffith et al. (1993) Neuron10:501-509).

An “arrhythmia” is a disturbance in the heart's natural rhythm.According to the present invention, the arrhythmia may be any atrial orventricular arrhythmia associated with a prolonged action potential,early afterdepolarizations, delayed afterdepolarizations, orintracellular calcium overload. Cardiac conditions which involve sucharrhythmias include, but are not limited to, long QT syndrome,cardiomyopathy, and the proarrhythmic effects of certain medicationsincluding antiarrhythmic drugs and ischemia. Other structuralabnormalities of the heart, abnormalities of the heart's electricalsystem, and diseases may also cause arrhythmias treatable by the presentinvention. One particular clinical indication for intervention is thepresence of prolonged QT intervals (>480 ms). The arrhythmia to betreated may be life-threatening. A preferred embodiment of the presentinvention is the treatment of ventricular tachycardia, an arrhythmiaassociated with early afterdepolarizations. Treatment of atrial andventricular fibrillation, arrhythmias associated with DADs andintracellular overload are also provided for by the invention.

A “sufficient amount” or an “effective amount” of a multifunctionalcalcium/calmodulin-dependent protein kinase inhibitor according to thisinvention is an amount sufficient to suppress early afterdepolarizations(EADs), delayed afterdepolarizations (DADs), or intracellular calciumoverload. Early afterdepolarizations are depolarizing oscillations inthe action potential and their likelihood of occurrence is increased bythe prolongation of action potential repolarization. They are also aprobable cause of some lethal arrhythmias. Delayed afterdepolarizationsare a marker of intracellular calcium overload and also the cause ofsome lethal arrhythmias.

An effective amount of inhibitor will vary with the activity of theinhibitor. In one embodiment of the invention, from about 0.05 to about5.0 mg of inhibitor per kilogram of subject's body weight isadministered. In a preferred embodiment, from about 0.3 to about 3.0 mgper kilogram is administered. In another embodiment, this dosage couldbe repeated every thirty to sixty minutes until suppression of earlyafterdepolarizations is achieved, unless limited by the development ofhypotension due to the Ca channel blocking effect. In anotherembodiment, a myristoylated CaM kinase inhibitory peptide isadministered at a dose of from about 2 to about 20 μmole per kilogram ofbody weight.

In one embodiment, the administration of a CaM kinase inhbitor may alsobe combined with administration of a second antiarrhythmic agent to thepatient. This combination may provide benefits both in terms of efficacyand safety. In a preferred embodiment, the CaM kinase inhibitor isadministered to the patient in conjunction with a second antiarrhythmicagent that is known to have a proarrhythmic effect. Existingantiarrhythmic agents which may be delivered to a patient in combinationwith the CaM kinase inhibitor include K-channel blockers, such as theclass IA or III antiarrhythmic drugs. In one embodiment of theinvention, the class IA agents procainamide (1.0 gram i.v. loading dosefollowed by 1-3 mg/min i.v.) or quinidine (˜70 mg/Kg/24 hours) would beadministered with a CaM kinase inhibitor. In another embodiment of theinvention, the class III agents dofetilide (8 mg/Kg i.v.) or d-sotalol(5.7 mg/Kg/24 hours p.o.) would be administered with a CaM kinaseinhibitor.

In another embodiment, the administration of a CaM kinase inhibitor mayalso be combined with administration of a β-adrenergic antagonist toincrease efficacy of antiarrhythmic therapy. In one embodiment of theinvention, the β-adrenergic antagonist atenolol (50-100 mg/dayp.o./i.v.), propranalol (60 mg/6 hours p.o. or 30 mg/6 hours i.v.), ormetoprolol (50-100 mg/6 hours p.o./i.v.) would be administered with aCaM kinase inhibitor.

In another embodiment, the administration of a CaM kinase inhibitor mayalso be combined with administration of a calcium channel blocker toincrease efficacy of antiarrhythmic therapy. In one embodiment of theinvention, the calcium channel antagonist nifedipine (10-30 mg/6 hoursp.o.), verapamil (80-120 mg/6 hours p.o. or 0.075-0.15 mg/Kg i.v.),diltiazem (30-90 μg/6 hours p.o. or 0.075-0.15 mg/Kg i.v.), mibefradil(50-100 mg/day p.o.) would be administered with a CaM kinase inhibitor.

In another embodiment of the invention, the administration of a CaMkinase inhibitor may be combined with a treatment that utilizes anantiarrhythmic device. This combination is useful in treating andpreventing arrhythmias. In a preferred embodiment, the antiarrhythmicdevice is an implantable cardiac defibrillator (also referred to hereinas an implantable cardioverter defibrillator). For instance, theadministration of a CaM kinase inhibitor may be combined with animplantable atrial cardiodefibrillator to treat atrial fibrillation. Inanother embodiment, the administration of a CaM kinase inhibitor may becombined with an implantable ventricular cardiodefibrillator to treatventricular fibrillation. In still another embodiment, theadministration of a CaM kinase inhibitor may be combined with apermanent pacing system to prevent atrial fibrillation.

In another embodiment, the present invention provides for apharmaceutical composition of a calcium/calmodulin-dependent proteinkinase inhibitor and a pharmaceutically acceptable excipient. In apreferred composition, an inhibitor of CaM kinase II is used.

A “pharmaceutically acceptable excipient” or “carrier” is atherapeutically inert substance which serves as a diluent or deliveryvehicle for the inhibitor drug. Selection of suitable carriers for thepharmaceutical composition is readily achievable by one skilled in theart and would in part be dictated by the specific CaM kinase inhibitorchosen. Typical pharmaceutical compositions useful for CaM kinase IIinhibitors are disclosed in Remington, The Science and Practice ofPharmacy, 19th ed., 1995, Mack Publishing Co., Easton, Pa., thedisclosure of which is incorporated by reference herein. Possiblepharmaceutical excipients include polymers, resins, plasticizers,fillers, binders, lubricants, glidants, disintegrants, solvents,co-solvents, buffer systems, surfactants, preservatives, sweeteningagents, flavoring agents, pharmaceutical grade dyes or pigments, andviscosity agents.

A method for identifying agents useful in the treatment or prevention ofarrhythmias is also provided by the present invention. Screening fornovel antiarrhythmia agents may be conducted by determining if the agentinhibits CaM kinase II activity. One test for CaM kinase II inhibitionis described herein; however, alternative methodologies for identifyinginhibitors via exposure to CaM kinase II are within the purview of thisinvention. The test for CaM kinase II inhibition may be done in vivo,but is preferably done in vitro. The ability of an assayed compound toinhibit the activity of CaM kinase II to an appreciable degree indicatesthat the compound will have antiarrhythmic properties.

The invention also provides for methods of suppressing early and delayedafterdepolarizations and intracellular calcium overload in a mammal.This suppression is achieved by the administration of an inhibitor of amultifunctional calcium/calmodulin-dependent protein kinase to thesubject. In a preferred embodiment, the inhibitor is a CaM kinase IIinhibitor.

Another aspect of the invention provides for methods of treatingarrhythmia in a human by decreasing the activity of a CaM kinase in thehuman. This decrease in activity may be achieved through a number ofways, each readily discernible by one skilled in the art. In oneembodiment, the activity of CaM kinase is decreased by theadministration of a CaM kinase inhibitor to the subject. In otherembodiments, the activity of a CaM kinase may be achieved through othermeans such as by decreasing the level of expression of a CaM kinase in ahuman by an antisense strategy or similar method.

The present invention, in all aspects, is enabled by our discovery thatEADs and DADs are due to I_(Ca) and/or intracellular calcium overloadand are facilitated by a multifunctional calcium/calmodulin-dependentprotein kinase.

In our experiments, EADs were monitored with monophasic action potential(MAP) catheters. Reliable EAD induction was achieved using hypokalemia,bradycardia, and the Vaughn-Williams class III antiarrhythmic agentclofilium in isolated perfused rabbit hearts. We first showed a role forI_(Ca) in clofilium-induced EADs using the specific I_(Ca) antagonistsnifedipine and Cd²⁺ (Example 1). EAD termination by I_(Ca) antagonistsappeared to be a direct effect of I_(Ca) blockade, and not a secondaryeffect of [Ca²⁺]_(i) depletion, because of its 1) rapid onset (FIGS. 1)and 2) occurance before significant loss of LVDP (FIG. 2). The lack ofMAP shortening at EAD termination is further evidence that EADtermination is a direct effect of I_(Ca) blockade and not due to asecondary effect on MAP duration (FIG. 3).

Further studies in isolated rabbit hearts examined the role of CaMkinase in facilitating EADs using the active CaM kinase inhibitor KN-93.Parallel experiments with the inactive analog KN-92 served as controls(Examples 2 and 3, FIGS. 1-3). Our experiments showed that the CaMkinase inhibitory drug KN-93, but not its inactive analog, KN-92,significantly decreased the inducibility of EADs in the whole heartmodel independent of other factors known to promote EADs. Actionpotential duration was not different in hearts pretreated with KN-92 orKN-93 before or after clofilium (FIG. 3). Similarly, heart rates in theKN-92 and KN-93 pretreated hearts were not different (FIG. 2). Wholecell mode voltage clamp experiments with isolated rabbit ventricularmyocytes established that KN-93 and KN-92 are equipotent direct blockersof I_(Ca) at the concentration (0.5 μmol/l) used in the isolated heartexperiment (Example 6, FIG. 6). However, ventricular CaM kinase activitywas significantly inhibited by KN-93 (K_(i)≦2.58 □mol/l), but not byKN-92, based on the results of an in vitro assay (Example 5, FIG. 5).

Corroborating results were obtained with a second CaM kinase inhibitor,a highly specific pseudo-substrate peptide, and an appropriate control(Example 7). The specificity of the peptides used excludes thepossibility of PKA and PKC being important mediators of arrhythmogeniccalcium current during these EADs.

The clofilium-induced increase in action potential duration is expectedto increase Ca²⁺ entry via I_(Ca). In addition to mediating Ca²⁺enhancement of I_(Ca), CaM kinase interacts with other [Ca²⁺]_(i)dependent processes in ventricular myocytes. CaM kinase both facilitatesCa²⁺ uptake by the sarcoplasmic reticulum and release by the ryanodinereceptor. The anticipated net effect of CaM kinase activation inventricular myocytes is increased LVDP. Clofilium did increase LVDP inKN-92 but not KN-93 pretreated hearts (FIG. 2). Once activated by[Ca²⁺]_(i), CaM kinase II undergoes intersubunit autophosphorylationwhich results in Ca²⁺-independent CaM kinase II activity. Measurement ofCa²⁺-independent activity by CaM kinase, thus, provides an importantmeasure of CaM kinase activation. We found that Ca²⁺-independentactivity is significantly increased during EADs and this increase isprevented by pretreatment with the CaM kinase inhibitor KN-93 (Example4, FIG. 4) at the same concentration found to suppress EADs.

In an additional set of experiments we applied repetitive stimulation ofisolated heart cells using voltage clamp with a prolonged actionpotential wave form (Example 8, FIG. 7). Inward currents consistentlydeveloped in cells treated with an inactive control peptide (FIG. 7B).These inward currents underlie delayed afterdepolarizations and areknown to result from intracellular calcium overload (Thandroyen et al.(1991) Circ Res 69:810-819). Inward currents were prevented in all cellstreated with a specific CaM kinase inhibitory peptide (Example 8, FIG.7C). Ryanodine, an agent that prevents intracellular calcium overload byblocking release of calcium from intracellular stores, also preventeddevelopment of the inward current in a separate group of cells. Thus,CaM kinase inhibition is effective in preventing inward currents knownto both report intracellular calcium overload and underlie DADs.

These results support the hypothesis that CaM kinase is activated duringEADs. The findings that 1) CaM kinase inhibition reduces EADinducibility and 2) EADs and MAP duration prolongation increase CaMkinase activity together suggest that CaM kinase is a proarrhythmicpositive feedback effector for EADs. CaM kinase inhibition alsosuppressed inward currents associated with DADs and intracellularcalcium overload. Therefore, we have established CaM kinase to be anovel antiarrhythmic drug target.

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

EXAMPLES Example 1

Clofilium-Induced Early Afterdepolarizations are Terminated by L-TypeCa²⁺ Current Antagonists

The experimental design for the isolated heart experiments is shown inFIG. 1A. All clofilium-induced EADs were terminated by the I_(Ca)antagonists Cd²⁺ (200-500 μmol/l) or nifedipine (10 μmol/l). EADtermination occured in <10 sec from a discernable decrease (>10% frompreceeding baseline) in LVDP after addition of I_(Ca) antagonist to theperfusate. EAD termination occurred before LVDP decreased significantly(FIG. 2A) and without a change in the interbeat interval (FIG. 2B).Action potential duration was not shortened upon the first beat of EADtermination. EAD termination by I_(Ca) antagonists was unlikely due to asecondary effect on [Ca²⁺]_(i) because LVDP was not significantlydepressed at initial termination. EAD termination by I_(Ca) antagonistswas not due to secondary effect on MAP duration or heart rate. Theseresults support the hypothesis that clofilium-induced EADs are directlydue to I_(Ca).

Experimental Methods

New Zealand White rabbits of either gender (2.7-3.2 kg) were treatedwith a heparin bolus (150 U/kg IV) and killed by pentobarbital (50 mg/kgIV) overdose. Hearts were rapidly excised and placed in ice-coldTyrode's solution for dissection of extracardiac tissue. (Tyrode'ssolution was composed of (mmol/l) NaCl (130.0), NaHCO₃ (20.0), glucose(5.6), KCl (3.0), CaCl₂ (2.0), NaH₂PO₄ (1.8), MgCl₂4H₂O(0.7).) The aortawas cannulated, and perfused retrograde with Tyrode's solution. Theperfusate (200 ml) was recirculated through a warming bath and fillingpressure was adjusted to 20 cm of H₂O. Recirculation time was ˜1 min,based on the time to decrease in left ventricular (LV) pressurefollowing addition of I_(Ca) blockers to the recirculating perfusate.Temperature was monitored using a thermistor probe positioned in theright ventricle (RV) and maintained at 33±1° C. by a closed loop feedback system. The perfusate was vigorously bubbled with 95% O₂, 5% CO₂and pH was monitored throughout the experiments and adjusted to 7.4 withLN NaOH or HCl, as appropriate. Both atria were removed and drains wereplaced in the RV and LV apices. An electrocardiogram (ecg) lead wassutured to the LV apex and the proximal pulmonary artery. Epicardialpacing wires were inserted into the RV free wall. Bradycardia wasproduced by crushing the AV node with a forceps. Epicardial pacingmaintained a minimum cycle length of 1500 ms (heart rate of 40beats/min). Pacing output was adjusted to twice diastolic threshold. Inmost experiments LV pressure was continuously monitored with a solidstate transducer (Camino Laboratories) placed in a saline filled latexballoon. The balloon was secured with a purse string suture around theleft atrial remnant and pressure was adjusted to maximize the LVdeveloped pressure (LVDP) keeping the LV end diastolic pressure <10mmHg. LVDP (peak systolic pressure-peak diastolic pressure) measurementsare reported as the mean of 3 contiguous beats except during irregularrhythms and at EAD termination. During irregular rhythms LVDPmeasurement was reported as the mean of 3 beats following the longestinterbeat intervals. LVDP is reported as the first beat without an EADfollowing I_(Ca) blockade (FIG. 1C). LVDP stability was defined as <10%interbeat variability.

In most experiments a single MAP electrode (EP technologies Inc.,Sunnyvale, Calif.) was positioned over the LV epicardium with amacro-manipulator. The heart was positioned against an adjustable stopopposite the epicardial MAP catheter to minimize heart motion. In apreliminary series of experiments designed to test the concordance ofendocardial and epicardial EADs the LV balloon was omitted and pairedMAP catheters were positioned opposite one another on the LV epicardialand endocardial free wall. MAP recordings were concordant for thepresence and absence of EADs in all but one instance, suggesting thatepicardial MAP recording is a valid method for detecting EADs from LVepicardium and endocardium in this isolated heart model. All MAP signalswere amplified from (0 to 100 Hz) with a direct current coupledpreamplifier (EP technologies Inc., Sunnyvale, Calif.) and recordeddirectly onto a chart recorder (Gould 2600S) at 100 mm/sec. Data fromsome experiments were digitized (Neuro-corder) and stored on a videotaperecorder. Acceptable MAP catheter position was confirmed by a stableaction potential configuration free of EADs or delayed afterdepolarizations (DADs) during a >10 minute control period. The MAPamplitude was defined as the difference between phase 2 and phase 4. MAPamplitude at the start of the experiments was 10.4(±1.26) mV. The MAPduration was measured from the onset of phase 0 to the point of 50%(APD₅₀) and 90% (APD₉₀) repolarization. When present, EADs were includedin the APD measurements. All MAP durations are reported as the mean of 3contiguous beats, except following EAD termination where the first beatafter EAD termination by Cd²⁺ or nifedipine was used. Beat to beatintervals were measured from the onset of phase 0 using contiguous MAPs.Beat to beat intervals during irregular rhythms are reported as thelongest intervals present during the measurement period.

EADs were defined as discrete oscillations in MAP repolarization withthe slope of the tangent to the onset of the oscillation >0.0 (FIG. 1).EADs had to be present on most (>90%) beats over a 1 min period or in astable bigeminal alternating pattern to be classified as inducible. Oncethese criteria were met EADs continued for >2 min and always requiredI_(Ca) blockade for termination. Multiple EADs were defined as >1 EADocurring per beat (FIG. 1). The CaM kinase inhibition experimentsconsisted of 4 periods (FIG. 1A). The first period (>10 min) was thecontrol and established stable baseline values for MAPs, LVDP, heartrate, and rhythm. The second period followed addition of KN-92 or KN-93to the perfusate and lasted 10 min. The third period followed additionof clofilium to the perfusate and lasted for 30 min or until EADsoccurred. If no EADs occured then the longest MAP durations were used.MAP and LVDP measurements were made at 10 min intervals and at the timeof EAD occurrence during the third period. The fourth period onlyconsisted of experiments where prior EADs occured, and measurementsfollowed addition of nifedipine (10 μmol/l) or Cd²⁺ (200-500 μmol/l) tothe perfusate.

Example 2

Early Afterdepolarizations are Prevented by Pretreatment with the CaMkinase Inhibitor KN-93

We investigated if CaM kinase contributed to ventricular EAD initiationand maintenance, given its role in the enhancement of I_(Ca). This wastested using the CaM kinase inhibitor KN-93, and its inactive analogKN-92. The decision to use KN-93 and KN-92 at 0.5 μmol/l was based onour finding that these agents were equally effective direct I_(Ca)antagonists at this concentration (FIG. 6C).

Isolated hearts pretreated with the the CaM kinase inhibitor KN-93 (0.5μmol/l) were significantly less likely to develop EADs in response toclofilium (7.5 μM) (EADs in 4/10 hearts) than hearts pretreated with theinactive analog KN-92 (EADs in 10/11 hearts) (0.5 μmol/l) (P=0.024). EADinduction by clofilium was not different in hearts pretreated with KN-92(EADs in 10/11 hearts) compared to hearts without any type ofpretreatment (EADs in 8/8 hearts), suggesting that the inactive analogKN-92 had no effect on EAD inducibility by clofilium (data not shown).When EADs were subclassified as single or multiple oscillations duringMAP repolarization, multiple EADs occurred more frequently in heartspretreated with the inactive analog KN-92 (7/11) compared with thosepretreated with the CaM kinase inhibitor KN-93 (1/10) (p=0.024). Thedifferences in EAD inducibility in hearts pretreated with KN-93 andKN-92 were not due to differences in MAP duration (FIG. 3) or heart rate(FIG. 2). These findings suggest that KN-93 suppression of EADs isindependent of known factors that facilitate EADs, such as heart rateand action potential duration.

Example 3

CaM Kinase Inhibition Prevented Increased Left Ventricular DevelopedPressure During Action Potential Prolongation and EarlyAfterdepolarizations

Left ventricular developed pressure increased significantly, comparedwith control, following addition of clofilium to the bath in heartspretreated with the inactive analog KN-92 (FIG. 2A). Left ventriculardeveloped pressure increases occurred in step with MAP durationprolongation (FIG. 3) and EADs, but without a change in heart rate (FIG.2B). KN-93 pretreatment completely prevented the increase in LVDP byclofilium (FIG. 2A) without reducing MAP duration prolongation (FIG. 3)or changing heart rate (FIG. 2B). Increased LVDP during MAP prolongationand EADs is likely due to CaM kinase-mediated I_(Ca) augmentation.

Example 4

Ca²⁺-Independent CaM Kinase Activity is Increased During EarlyAfterdepolarizations

Once activated by increased [Ca²⁺]_(i) CaM kinase II activity becomesCa²⁺-independent via intersubunit phosphorylation. Thus,Ca²⁺-independent CaM kinase activity is a marker for CaM kinaseactivation. Because EADs appear due to I_(Ca) in this isolated heartmodel, we hypothesized that EADs are associated with increasedCa²⁺-independent CaM kinase activity. Ca²⁺-independent CaM kinaseactivity increased significantly in hearts demonstrating EADs afterclofilium compared to hearts without clofilium treatment or EADs (FIG.4). No significant increase in Ca²⁺-independent CaM kinase activityoccurred, compared to control, in hearts pretreated with KN-93 (0.5μmol/l) prior to clofilium (FIG. 4). These findings show that EADs areassociated with an increase in Ca²⁺-independent CaM kinase activity andthat the concentration of KN-93 used to suppress EADs in theseexperiments is sufficient to significantly inhibit CaM kinaseactivation. This increase in Ca²⁺-independent CaM kinase activity isconsistent with the hypothesis that CaM kinase is a proarrhythmicsignaling molecule for EADs.

Example 5

KN-93 Inhibits CaM Kinase Activity in Rabbit Myocardium

In vitro measurements of CaM kinase activity in LV tissue homogenateshow that KN-93 is an inhibitor of CaM kinase with a K_(i)<2.58 μmol/l(FIG. 5). The inactive analog KN-92 did not cause appreciable inhibitionof CaM kinase activity. These findings are similar to earlier findingsreported in rat pheochromocytoma (PC12) cells. These results show thatKN-92 is a valid control for CaM kinase inhibition by KN-93.

Experimental Methods

Left ventricular homogenate was prepared as follows: after removal ofthe heart, it was placed in nominally Ca²⁺ free ice-cold HEPES-bufferedTyrode's solution. One to two grams of the left ventricular free wallwas cut away, coarsely minced, and suspended in 5-10 ml of coldhomogenization buffer (mmol/l: 20 PIPES, 1 EDTA, 1 EGTA, 2 DTT, 10sodium pyrophosphate, and 50-100 μg/ml leupeptin) at pH 7.0. Tissuesuspension was homogenized at 4° C. using 3-10 sec bursts of a Polytronwith 30 sec pauses inbetween bursts. The homogenate was then centrifugedat ˜10,000×g for 20 min at 4° C. using a JA-20 rotor. The supemate wasremoved and used directly in the assay. Protein concentration wasmeasured by the method of Bradford, using BSA as the standard.

Assay of CaM kinase activity was performed essentially as described byWaldmann et al. with a few modifications. The assay was performed intriplicate in a final volume of 50 μl containing 50 mmol/l PIPES, pH7.0, 10 mmol/l MgC12, 0.1 mg/ml BSA, 10 μmol/l autocamtide 3 (synthetickinase substrate), 150 nmol/l calmodulin, 1 mmol/l CaCl₂ or 1 mmol/lEGTA, and increasing concentrations (0-100 μmol/l) of KN-93 or KN-92.30-50 μg of tissue homogenate were added per assay tube, and sampleswere then pre-incubated at 30° C. for 1 min. The kinase reaction wasthen started by addition of 50 μmol/l (final) γ³²P-ATP (˜400 cpm/pmol),and incubation was carried out for an additional 1 to 2 minutes. Thereaction was terminated by addition of 10 μl cold trichloroacetic acid(30% w/v), and samples were then placed on ice for ≧2 min.Calcium/calmodulin-dependent phosphorylation of the autocamtide 3substrate was then quantitated as described.Calcium/calmodulin-independent CaM kinase activity was quantitated asdescribed above, but Ca²⁺ was omitted from the assay solution. Datadescribing inhibition of CaM kinase activity by KN-93 was fit accordingto the Hill equation.

Example 6

KN-92 and KN-93 are Equipotent Direct Blockers of L-Type Ca²⁺ Current

We assessed the relative L-type Ca²⁺ current (I_(Ca)) blocking potencyof KN-92 and KN-93 in single ventricular myocytes using whole cellvoltage clamp methodology. These studies were performed because of theobservation that clofilium-induced EADs are terminated by I_(Ca)blockade in isolated rabbit hearts, along with findings that other CaMkinase inhibitory agents block I_(Ca). Since CaM kinase is known toenhance I_(Ca), we attempted to block CaM kinase activation by dialyzingcells, via the recording micropipette, with the fast Ca²⁺ chelator BAPTA(20 mmol/l), so that only direct effects of KN-92 and KN-93 on I_(Ca)were measured. External application of either KN-92 (0.5 μmol/l) orKN-93 (0.5 μmol/l) caused equipotent steady state inhibition of peakI_(Ca) (36.2±1.9%, KN-92 and 36.2±4.3%, KN-93) in isolated rabbitventricular myocytes (FIG. 5). Estimated IC₅₀ values were ˜μmol/l forboth drugs. Neither KN-92 or KN-93 shifted the apparent voltagedependence of I_(Ca) activation. The amount of I_(Ca) inhibition by 0.5μmol/l KN-92 or KN-93 is apparently insufficient to suppress EADs inthis model (Example 2). These results show that KN-92 is a valid controlfor the direct I_(Ca) blocking effects of KN-93 in the isolated rabbitventricular myocytes because these agents have equivalent I_(Ca)antagonist effects at the concentration (0.5 □mole/l) used to suppressEADs (FIG. 6C).

Experimental Methods

Single ventricular myocytes were prepared as previously described inAnderson et al. (1994) Circ Res, 75:854-861. The standard Ca²⁺containing solution for myocyte preparation was composed of (mmol/l)NaCl 137.0, HEPES (free acid) 10.0, NaH₂PO₄ 0.33, glucose 10.0, KCl 5.4,CaCl₂ 1.8, and MgCl₂ 2.0. The nominally Ca²⁺ free solution wasidentical, except CaCl₂ was omitted. The low Ca²⁺ solution contained 0.2mmol/l CaCl₂. The collagenase solutions were prepared with 1% BSA(wt/vol) and ˜60 U/ml collagenase (Worthington Biochemicals) and ˜0.1U/ml type XIV protease (Sigma Chemical Co). The myocyte bath solutioncontained (mmol/1) NaCl 137.0, CsCl 20.0, glucose 10.0, HEPES 10.0, KCl5.4, CaCl₂ 1.8, MgCl₂ 0.5, and tetrodotoxin 0.03; pH was adjusted to 7.4with 10N NaOH. The intracellular pipette solution contained (mmol/l)CsCl 130.0, 4Cs BAPTA 10.0 (Molecular Probes), phosphocreatine 5.0,NaGTP 1.0, and MgATP1.0; pH was adjusted to 7.2 with 10N CsOH.

New Zealand White rabbits (2 to 3 kg body weight) were killed bypentobarbital (50 mg/kg IV) overdose. Hearts were rapidly excised andplaced in ice cold nominally Ca²⁺ free HEPES-buffered myocyte bathsolution. The aorta was cannulated, and the heart was perfused in aretrograde fashion with a nominally Ca²⁺ free perfusate for 15 minutesat 37° C. This was followed by a 15-min perfusion withcollagenase-containing nominally Ca²⁺ free solution. Final perfusion waswith collagenase-containing low Ca²⁺ (0.2 mmol/L) solution. The LV andseptum were cut away, coarsely minced, and placed in a beaker containinglow Ca²⁺ solution with 1% (wt/vol) bovine serum albumin (BSA) at 37° C.Myocytes were dispersed by gentle agitation, collected in serialaliquots, and then maintained in standard saline solution containing 1.8mmol/L Ca²⁺. All solutions were vigorously oxygenated.

Isolated quiescent ventricular myocytes were studied with patch-clampmethodology in the whole cell recording configuration by using anAxopatch 1B amplifier (Axon Instruments). Micropipettes were pulled fromglass capillary tubing (VWR) and heat-polished to a tip resistance of1-3 MΩ when filled with the intracellular solution.

The cell membrane was commanded from −40 mV to +60 mV for 200 ms in 10mV steps from a holding potential of −40 mV at a stimulation of 1.0 Hz.This protocol was repeated with 60 sec pauses between series of voltageclamp steps, and transmembrane current was sampled at 2 kHz. Voltageclamp protocols and data acquisition were performed using PCLAMPsoftware (version 6.0, Axon Instruments) on a microcomputer (Gateway2000) with a A/D, D/A convertor (Digidata 1200, Axon Instruments). Allexperiments were performed at room temperature (20° C. to 23° C.) on thestage of a Diaphot inverted microscope (Nikon corp). Pure macroscopicI_(Ca) is observed under these recording conditions. Rapid intracellularCa²⁺ buffering with BAPTA (10 mmol/l) and the long (60 sec) restsbetween voltage step protocols were chosen to minimize CaM kinaseactivation so direct I_(Ca) blockade by KN-93 or KN-92 was measured,independent of an effect on CaM kinase. Peak I_(Ca) stability wasdefined when peak I_(Ca) varied <10% between voltage steps. DirectI_(Ca) blockade by KN-92 or KN-93 was examined after a stable controlperiod. KN-92 or KN-93 was added to the bath at increasingconcentrations. Peak I_(Ca) stability was established before changing tothe next KN-92 or KN-93 concentration.

Example 7

Early Afterdepolarizations are Also Prevented by Pretreatment withHighly Specific CaM Kinase Inhibitory Peptides

Experiments performed in isolated rabbit heart cells using highlyspecific CaM kinase inhibitory peptides further corroborated the resultsobtained using the inhibitor KN-93 (Examples 2 and 3). The peptide ofthe sequence KKALHRQEAVDCL (SEQ ID NO: 1) had been previously reportedto inhibit CaM kinase, but not PKA or PKC. A second peptide,KKALHAQERVDCL (SEQ ID NO: 2), which does not inhibit CaM kinase was usedas a control.

Action potentials were induced in isolated rabbit ventricular myocytesstudied in current clamp with low (1.0 mM) extracellular potassium tofavor EAD induction. EADs were induced in the majority of control cellsdialyzed with an inactive peptide (11/15) but not in cells dialyzed withthe CaM kinase inhibitory peptide (4/15). EADs seen in cells treatedwith the control peptide tended to be complex and repetitive (i.e.multiple), but only single EADs were present in inhibitory peptidetreated cells.

Furthermore, measurements of the L-type calcium window current using aprolonged EAD-containing action potential as a voltage clamp wave formrevealed that an increase in the current was prevented by dialysis withthe CaM kinase inhibitory peptide (data not shown). The control peptidehad no significant effect on this calcium current. These results areconsistent with the augmentation of arrhythmogenic L-type calcium windowcurrent by CaM kinase.

Example 8

Inward Currents Associated with Delayed Afterdepolarizations andIntracellular Calcium Overload are Suppressed by CaM Kinase Inhibition

Isolated heart cells (Experimental Methods, Example 6) were stimulated(0.5 Hz) in voltage clamp using a prolonged action potential wave form(FIG. 7A). Rapid pacing favors intracellular calcium overload. Inwardcurrents that develop in response to rapid pacing are a marker forintracellular calcium overload and they are the basis for arrhythmogenicdelayed afterdepolarizations (DADs). The majority of cells (5/6) treatedwith an inactive control peptide (SEQ ID NO: 2) developed inwardcurrents (FIG. 7B). Treatment of a second group with the active CaMkinase inhibitory peptide (SEQ ID NO: 1) completely prevented the inwardcurrents (inward currents in 0/5 cells) (FIG. 7C). Ryanodine (10μmole/l), prevents intracellular calcium overload by blocking release ofcalcium from intracellular stores, also prevented development of pacinginduced inward currents (in 2/2 cells tested). These results indicatethat CaM kinase inhibition can prevent intracellular calcium overloadand development of DAD-linked currents.

Experimental Methods

Action potential recording was performed in the following bath solution(mmol/L): NaCl 140.0, Glucose 10.0, HEPES 5.0, KCl 5.4, CaCl₂ 2.5, MgCl₂1.0; pH was adjusted to 7.4 with 10 N NaOH. The intracellular pipettesolution for action potential recording contained (mmol/L): K aspartate120.0, HEPES 10.0, EGTA 10.0, Na₂ATP 5.0, MgCl₂ 4.0, CaCl₂ 3.0; pH wasadjusted to 7.2 with 1N KOH. Unless otherwise noted, all chemicals werefrom Sigma.

Cells were stimulated at 0.1 Hz in whole cell configuration in currentclamp mode with 0.1 to 1.0 nA pulses of depolarizing current(1.25×threshold) for 2-3 ms at room temperature (20°-23° C.). Actionpotentials were low pass filtered at 2 KHz and sampled at 2.5 KHz with a12-bit analog to digital converter (Digidata 1200 B, Axon Instruments).A long action potential waveform was digitized and stored forapplication as a voltage command using pClamp 6.03.

Isolated cells were studied with the action potential command wave formusing voltage clamp methodology (Experimental Methods, Example 6). Cellswere stimulated at 0.5 Hz and 32° C. (heated stage from WarnerInstruments) for up to 100 beats. Recording solutions were identical tothose for action potential recording except that the calcium buffer EGTAwas omitted from the intracellular solution to favor intracellularcalcium overload. The presence or absence of the post-repolarizationinward current was noted.

All documents cited in the above specification are herein incorporatedby reference. Various modifications and variations of the presentinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for identifying agents that inhibitcalcium/calmodulin-dependent kinase II (CaM kinase II) and are usefulfor treating arrhythmias, the method comprising: (a) contactingCaMkinase II with an agent; (b) assaying the activity of the contacted CaMkinase II in vitro; and if CaM kinase II activity is reduced, then (c)administering said agent to an isolated heart cell, an isolated heart,or a test mammal; and then (d) detecting the presence or absence ofarrhythmia; wherein a reduction of arrhythmia indicates that said agentis an inhibitor of CaM kinase II that is useful for treatingarrhythmias.
 2. The method of claim 1, wherein the assaying stepcomprises adding autocamtide-3 to the CaM kinase II in vitro andmeasuring phosphorylation of said autocamtide-3 by CaM kinase II.
 3. Themethod of claim 1, wherein arrhythmia is detected in the test mammalusing an electrocardiogram (ECG).
 4. The method of claim 1, wherein thetest mammal is a rabbit.
 5. The method of claim 1, wherein arrhythmia isdetected by measuring monophasic action potentials.